Gastrointestinal (GI) bacteria sense diverse environmental signals, including host hormones and nutrients, as cues for differential gene regulation and niche adaptation. Although the impact of carbon nutrition on the colonization of the gut by the microbiota has been extensively studied, the extent to which carbon sources affect the regulation of virulence factors by invading pathogens has not been fully defined. The PI has shown that the enteric pathogen enterohemorrhagic Escherichia coli (EHEC) gages sugar sources as an important cue to regulate expression of its virulence genes. Specifically, this sugar dependent regulation fine tunes the expression of the locus of enterocyte effacement (LEE) pathogenicity island necessary for the formation of attaching and effacing (AE) lesions on enterocytes. Glycolytic environments inhibit the expression of the LEE genes. Conversely, growth within a gluconeogenic environment activates expression of these genes. Part of this sugar-dependent regulation is achieved through two transcription factors: KdpE and Cra. Cra and KdpE interact to optimally directly activate expression of the LEE genes in a metabolite dependent fashion. This sugar dependent regulation is key during infection of the mammalian host, given that a kdpE mutant is attenuated in vivo. Additionally, a novel two component system, named FusKR (where FusK is a membrane bound histidine sensor kinase (HK), and FusR a response regulator (RR)) that senses fucose, controls expression of the LEE genes. This fucose-sensing system is required for robust EHEC intestinal colonization. During growth in mucus, the glycophagic prominent member of the GI microbiota, Bacteroides thetaiotaomicron, supplies fucose to EHEC, modulating its virulence gene expression. However, several questions remain answered, such as how does the interplay among the KdpE, Cra and FusR transcription factors controls optimal expression of the LEE genes? It is also known that FusK senses fucose, but an extensive investigation on whether this sensor is responsive to other sugar sources is lacking. Additionally, it is unknown whether FusK exclusively phosphorylates its cognate RR FusR, or whether it can also phosphorylate other non-cognate RRs. Finally, the implications of these complex sugar sensing interactions during mammalian infection, although clearly important due to the attenuation of kdpE and fusR mutants in vivo, remains to be addressed. Hence, the Specific Aims of this grant are: Aim 1: Unravel the mechanistic interactions among KdpE, Cra and FusR on LEE gene expression. Aim 2: Investigate the FusK phosphorelay signaling cascade. Aim 3: Sugar regulation of virulence during mammalian infection.